Hydrophobic photolabeling in membranes: the human erythrocyte glucose transporter.
Human erythrocyte membranes were labeled with a hydrophobic photoactivable reagent, 2-[3H]Diazofluorene. Electrophoretic analysis of the protein fraction showed that several membrane spanning proteins like Band 3 (the anion transporter), Band 4.5 (the glucose transporter), and the sialoglycoproteins PAS 1, 2, and 3 have been labeled. To isolate the diazofluorene-labeled glucose transporter, the membrane preparation was solubilized with Triton X-100 and passed through a DEAE-cellulose column. The flow-through fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive analysis of the gel indicated that besides the Band 4.5, two more proteins corresponding to the Band 3 and Band 6 regions also coelute with the glucose transporter in the flow-through fraction. On the other hand, use of n-octyl glucoside gave a relatively better preparation. The 2-[3H]DAF-labeled glucose transporter isolated by the latter method on tryptic digestion indicated that the Mr 18,000 fragment corresponding to the C-terminal transmembrane fragment is labeled.
Lala
A K
AK
Department of Chemistry, Indian Institute of Technology Bombay, Powai, India.
Bhat
S
S
D000345:Affinity Labels; D003902:Detergents; D003979:Diazonium Compounds; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005449:Fluorenes; D005960:Glucosides; D006801:Humans; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification*; D009004:Monosaccharide Transport Proteins / Q000097:blood / Q000302:isolation & purification*; D017830:Octoxynol; D010446:Peptide Fragments / Q000302:isolation & purification; D011092:Polyethylene Glycols; D014316:Tritium; D014357:Trypsin
D000345:Affinity Labels; D003902:Detergents; D003979:Diazonium Compounds; D005449:Fluorenes; D005960:Glucosides; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D014316:Tritium; C018619:octyl-beta-D-glucoside; D011092:Polyethylene Glycols; C052770:2-diazofluorene; D017830:Octoxynol; D014357:Trypsin